1. Field of the Invention
This invention relates to a polypeptide which inhibits the replication of Herpes Simplex Virus (HSV) and like viruses and its therapeutic use against infections of such viruses.
2. Description of the Prior Art
HSV exists as several serotypes of which HSV-1 is one which is clinically significant in relation to cold sores. HSV-1 is a DNA virus which is transcribed and replicated within the cell nucleus. As with many other viruses, the genes are transcribed into mRNA at different times. Certain important genes which are the first to be transcribed are denoted immediate-early (IE) or ".alpha.". Their transcription is enabled by promoter sequences which lies to the 5' end or upstream of the ATG start codon of the gene. Further upstream from the promoter the IE genes have a distinctive nucleotide sequence which is a consensus (common) sequence TAATGARAT (where R=purine, i.e. can be G or A).
The IE genes of HSV are induced by a component of the virion, first identified by M. E. M. Campbell, J. W. Palfreyman and C. M. Preston, Journal of Molecular Biology 180, 1-19 (1984), as "Vmw 65". Vmw 65, which has also been referred to as VP16, is a tegument protein which lies between the viral membrane and the capsid. Vmw 65 is said to be "trans-acting" or "transactivating". This language indicates merely that it is some soluble factor which acts on the viral DNA to regulate it. Yet another name for Vmw 65 is the .alpha.-Trans Inducing Factor or .alpha.-TIF, meaning that it acts on the viral DNA to induce transcription of the IE (.alpha.) genes.
M. E. M. Campbell et al., loc. cit. speculated that Vmw 65 might bind to the DNA in the TAATGARAT region, either directly or indirectly by modifying a host cell polypeptide. Later work, beginning with that of T. M. Kristie and B. Roizman, Proc. Natl. Acad. Sci. USA, 84, 71-75 (1987), has shown that the TAATGARAT region, which is termed a "cis-acting site" or the .alpha.-Trans Induction Cis-acting (.alpha.-TIC) site, does not bind directly to Vmw 65, but does bind to one or more host cell proteins. Various groups of workers have identified host cell proteins which bind both to the TAATGARAT region and to Vmw 65. They have been variously designated as ".alpha.-H1", "HC3", "octamer-binding protein", "OTF-1" and "TAATGARAT Recognition Factor" (TRF), all of which are probably identical. The TRF nomenclature is used in this specification.
Current knowledge is summarised by C. I. Ace et al., J. Virology 63, 2260-2269 (May 1989). Vmw 65 indicates the induction of IE genes by associating with cellular proteins, to form a complex known as IEC or TRF.C, which is able to bind specifically to DNA sequences which include TAATGARAT. In other words, IE gene induction involves a complex which is at least ternary between, at least, Vmw 65, TRF and a TAATGARAT region sequence.
It would be desirable to block the formation of this complex and thereby block induction of IE gene transcription of HSV. C. I. Ace et al., supra, have demonstrated that a virus mutant which lacks the ability to form such a complex with Vmw 65 and TRF replicates very poorly at low multiplicity of infection (MOI). Low MOI would be encountered clinically. Attempts have therefore been made to identify regions of Vmw 65 responsible for complex formation. It might then be possible to synthesise a short polypeptide which would compete with Vmw 65 in the formation of the complex.
S. J. Triezenberg, R. C. Kingsbury and S. L. McKnight, Genes & Development 2, 718-729 (1988) explored Vmw 65 structure/function with an assay for IE gene transcriptional induction and for the ability of Vmw 65 deletion mutants to inhibit IE transciptional induction by normal Vmw 65. They showed that if the carboxy terminus of Vmw 65 was deleted, the protein would no longer induce IE transcription, but reported that this deleted protein could prevent IE induction by normal Vmw 65. Using various deleted forms of Vmw 65 they showed that the boundaries for this inhibitory activity (i.e. inhibition of normal Vmw 65 when the two are together) mapped at the N-terminus somewhere between amino acids 56 and 74 and at the C-terminus somewhere between 380 and 393. Since their proposition is that the competitive inhibitory activity is due to an interaction with a cellular intermediate, they claim that these boundaries may be the boundaries for interaction with this cellular intermediate. Note that the assay was for gene transcription, i.e. essentially for an "end product". Therefore, the inhibitory activity could actually take place at any of a wide range of steps, e.g. by saturating sites for transport of the virus into the nucleus of the cell.
G. Werstuck and J. P. Capone, Gene 75, 213-224 (1989), have also explored Vmw 65 structure and function using measurements of the expression of a cat gene linked to an IE promoter region as an assay for the transcriptional induction function of Vmw 65. They found a total loss of IE induction activity when 4 or 5 amino acids were inserted into the Vmw 65 coding sequence at amino acids 178, 215, 335, 369 or 471 or when Vmw 65 was deleted in any of the following regions: amino acids 26-140, 26-177, 26-240, 142-177, 174-240, 179-412, 242-412, 331-412 and 331-470. In addition, in a similar assay to that used by Triezenberg et al, they examined the ability of the deleted mutants of Vmw 65 to competitively inhibit the IE induction function of normal Vmw 65. Competitive inhibition was obtained with mutants deleted from 331-470, 331-412, 242-412 or 186-490, indicating that the boundary of this competitive inhibiting activity mapped at an amino acid lower than 186. It is noteworthy and illustrative of the complications resulting from using this sort of assay to attempt to relate structure of Vmw 65 to function, that the boundary mapped for competitive inhibitory activity by Werstuck and Capone differs substantially from that mapped by Triezenberg et al., supra.
R. Greaves and P. O'Hare, Journal of Virology 63, 1641-1650 (April 1989), directly demonstrated that the acidic C-terminal domain of Vmw 65 (from amino acids 403 to the C-terminus) is not required for complex formation but that within the sequence of amino acids 317-403 there is a region which is required for complex formation.
C. I. Ace et al., J. Gen. Virology 69, 2595-2605 (1988) and Journal of Virology 63, 2260-2269 (May 1989) have performed biochemical studies of DNA-protein complex formation. This group has shown that insertion of a linker encoding a small number of amino acids (usually 4), at any of several of positions in the Vmw 65 sequence, directly prevents the ability of these altered proteins to form a complex with TRF. Amongst these, an insertion at amino acid 379 prevented the ability to form complex. In parallel, they confirmed that those mutants unable to form complex with TRF were unable to induce IE expression.